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Background The vast and promising class of long non-coding RNAs (lncRNAs) has been under investigation for distinct therapeutic applications. Nevertheless, their role as molecular drivers of bone regeneration remains poorly studied. The lncRNA H19 mediates osteogenic differentiation of Mesenchymal Stem/Stromal Cells (MSCs) through the control of intracellular pathways. However, the effect of H19 on the extracellular matrix (ECM) components is still largely unknown. This research study was designed to decode the H19 -mediated ECM regulatory network, and to reveal how the decellularized siH19-engineered matrices influence MSC proliferation and fate. This is particularly relevant for diseases in which the ECM regulation and remodeling processes are disrupted, such as osteoporosis. Methods Mass spectrometry-based quantitative proteomics analysis was used to identify ECM components, after oligonucleotides delivery to osteoporosis-derived hMSCs. Moreover, qRT-PCR, immunofluorescence and proliferation, differentiation and apoptosis assays were performed. Engineered matrices were decellularized, characterized by atomic force microscopy and repopulated with hMSC and pre-adipocytes. Clinical bone samples were characterized by histomorphometry analysis. Results Our study provides an in-depth proteome-wide and matrisome-specific analysis of the ECM proteins controlled by the lncRNA H19 . Using bone marrow-isolated MSC from patients with osteoporosis, we identified fibrillin-1 (FBN1), vitronectin (VTN) and collagen triple helix repeat containing 1 (CTHRC1), among others, as having different pattern levels following H19 silencing. Decellularized siH19-engineered matrices are less dense and have a decreased collagen content compared with control matrices. Repopulation with naïve MSCs promotes a shift towards the adipogenic lineage in detriment of the osteogenic lineage and inhibits proliferation. In pre-adipocytes, these siH19-matrices enhance lipid droplets formation. Mechanistically, H19 is targeted by miR-29c, whose expression is decreased in osteoporotic bone clinical samples. Accordingly, miR-29c impacts MSC proliferation and collagen production, but does not influence ALP staining or mineralization, revealing that H19 silencing and miR-29c mimics have complementary but not overlapping functions. Conclusion Our data suggest H19 as a therapeutic target to engineer the bone ECM and to control cell behavior.

Osteoporosis is a systemic disease that results in loss of bone density and increased fracture risk, particularly in the vertebrae and the hip. This condition and associated morbidity and mortality increase with population ageing. Long noncoding (lnc) RNAs are transcripts longer than 200 nucleotides that are not translated into proteins, but play important regulatory roles in transcriptional and post-transcriptional regulation. Their contribution to disease onset and development is increasingly recognized. Herein, we present an integrative revision on the studies that implicate lncRNAs in osteoporosis and that support their potential use as therapeutic tools. Firstly, current evidence on lncRNAs involvement in cellular and molecular mechanisms linked to osteoporosis and its major complication, fragility fractures, is reviewed. We analyze evidence of their roles in osteogenesis, osteoclastogenesis, and bone fracture healing events from human and animal model studies. Secondly, the potential of lncRNAs alterations at genetic and transcriptomic level are discussed as osteoporosis risk factors and as new circulating biomarkers for diagnosis. Finally, we conclude debating the possibilities, persisting difficulties, and future prospects of using lncRNAs in the treatment of osteoporosis.

Background Pancreatic ductal adenocarcinoma (PDAC) is among the cancer types with poorest prognosis and survival rates primarily due to resistance to standard-of-care therapies, including gemcitabine (GEM) and olaparib. Particularly, wild-type (wt)BRCA tumours, the most prevalent in PDAC, are more resistant to DNA-targeting agents like olaparib, restraining their clinical application. Recently, we disclosed a monoterpene indole alkaloid derivative (BBIT20) as a new inhibitor of homologous recombination (HR) DNA repair with anticancer activity in breast and ovarian cancer. Since inhibition of DNA repair enhances the sensitivity of cancer cells to chemotherapy, we aimed to investigate the anticancer potential of BBIT20 against PDAC, particularly carrying wtBRCA. Methods In vitro and in vivo PDAC models, particularly human cell lines (including GEM-resistant PDAC cells), patient-derived organoids and xenograft mice of PDAC were used to evaluate the anticancer potential of BBIT20, alone and in combination with GEM or olaparib. Disruption of the BRCA1-BARD1 interaction by BBIT20 was assessed by co-immunoprecipitation, immunofluorescence and yeast two-hybrid assay. Results The potent antiproliferative activity of BBIT20, superior to olaparib, was demonstrated in PDAC cells regardless of BRCA status, by inducing cell cycle arrest, apoptosis, and DNA damage, while downregulating HR. The disruption of DNA double-strand breaks repair by BBIT20 was further reinforced by non-homologous end joining (NHEJ) suppression. The inhibition of BRCA1-BARD1 heterodimer by BBIT20 was demonstrated in PDAC cells and confirmed in a yeast two-hybrid assay. In GEM-resistant PDAC cells, BBIT20 showed potent antiproliferative, anti-migratory and anti-invasive activity, overcoming GEM resistance by inhibiting the multidrug resistance P-glycoprotein, upregulating the intracellular GEM-transporter ENT1, and downregulating GEM resistance-related microRNA-20a and GEM metabolism enzymes as RRM1/2. Furthermore, BBIT20 did not induce resistance in PDAC cells. It inhibited the growth of patient-derived PDAC organoids, by inducing apoptosis, repressing HR, and potentiating olaparib and GEM cytotoxicity. The enhancement of olaparib antitumor activity by BBIT20 was confirmed in xenograft mice of PDAC. Notably, it hindered tumour growth and liver metastasis formation, improving survival of orthotopic xenograft mice of PDAC. Furthermore, its potential as a stroma-targeting agent, reducing fibrotic extracellular matrix and overcoming desmoplasia, associated with an enhancement of immune cell response by depleting PD-L1 expression in tumour tissues, renders BBIT20 even more appealing for combination therapy, particularly with immunotherapy. Conclusion These findings underscore the great potential of BBIT20 as a novel multifaceted anticancer drug candidate for PDAC treatment.

Osteoclasts are the only cells able to resorb all the constituents of the bone matrix. While the modulation of osteoclast activity is well established for preventing bone-related diseases, there is an increasing demand for novel classes of anti-resorption agents. Herein, we investigated non-coding RNA molecules and proposed DLEU1 and miR-16 as potential candidates for modulating osteoclast functions. DLEU1 and miR-16 target cell fusion at both the early and late stages of osteoclastogenesis but operate through independent pathways. DLEU1 silencing hinders the fusion process, leading to abrogation of the phagocytic cup fusion modality and a reduction in the fusion events between mononucleated precursors and multinucleated osteoclasts, while miR-16 influences monocyte-to-osteoclast differentiation, impairing osteoclasts formation but not the number of nuclei at early stages. On the other hand, using these non-coding RNAs to engineer mature osteoclasts has implications for bone resorption. Both DLEU1 and miR-16 influence the speed of resorption in pit-forming osteoclasts, without affecting the resorbed area. However, the impact of increasing miR-16 levels extends more broadly, affecting trench-forming osteoclasts as well, leading to a reduction in their percentage, speed, and resorbed area. These findings offer potential new therapeutic targets to ameliorate bone destruction in skeletal diseases.

The interplay between osteoblasts and osteoclasts (OCs) is a highly regulated and coordinated process essential for maintaining bone skeletal integrity and health. Disruption of this balance marks the onset of various bone disorders, such as osteoporosis. In our previous study, we demonstrated that non-coding RNAs (ncRNAs) were able to regulate OC behaviour. Specifically, the long ncRNA DLEU1 and the small ncRNA miR-16 independently control OCs multinuclearity, fusion and resorption. Herein, we investigate whether OCs modulated with ncRNAs impact the behaviour of osteoblast lineage cells (OBs) in a biomimetic co-culture system. Communication between OB and OC comprises numerous molecules and processes, including the secretion of coupling factors, release of matrix-derived signals during bone resorption, and exposure of previously occluded proteins within the bone resorption cavities. Firstly, we explored how ncRNAs influence the composition of the secretome and the remodelling of resorption cavities left by the ncRNA-engineered-OCs on OBs responses. In our OC/OB co-culture system, conducted in the absence of FBS and exogenous RANKL, we observed changes in the levels of secreted factors 72 h after DLEU1 levels were decreased in mature OCs (siDLEU1-OC condition), specifically in RANKL, leptin, osteocalcin, MMP7, MMP8, TIMP1 and TIMP2. Meanwhile, leptin and MMP8 levels were increased in the OC/OB co-culture when miR-16 was overexpressed in OCs (miR-16-OC condition). The results also show that OBs co-cultured with siDLEU1-OCs exhibited impaired migratory capacity and increased ALP levels, while OBs co-cultured with miR-16-OCs displayed a tendency towards decreased elongation. On bone slices, OBs persisted for a longer duration in the trenches created by siDLEU1-OCs. Besides altering OBs behaviour, silencing of DLEU1 reduced the area of each individual trench cavity, while miR-16 overexpression significantly hindered the total eroded surface and inhibited trench formation in the OC/OB co-culture system. Overall, these findings identify DLEU1 and miR-16 as key regulators in the interplay between human primary OBs and OCs, highlighting the potential of targeting ncRNAs in OCs to modulate their paracrine and matrix-mediated effects on OBs.

Multiple myeloma (MM) is the second most frequent hematological disease and can cause skeletal osteolytic lesions. This study aims to evaluate the expression of circulating microRNAs (miRNAs) in MM patients and to correlate those levels with clinicopathological features, including bone lesions. A panel of miRNAs associated with MM onset and progression, or with bone remodeling, was analyzed in the plasma of 82 subjects (47 MM patients; 35 healthy controls). Results show that miR-16-5p, miR-20a-5p, and miR-21-5p are differently expressed between MM patients and healthy controls. Receiver operating characteristic analyses indicate that their combined expression has potential as a molecular marker (Area Under the Curve, AUC of 0.8249). Furthermore, significant correlations were found between the analyzed miRNAs and disease stage, treatment, β2 microglobulin, serum albumin and creatinine levels, but not with calcium levels or genetic alterations. In this cohort, 65.96% of MM patients had bone lesions, the majority of which were in the vertebrae. Additionally, miR-29c-3p was decreased in patients with osteolytic lesions compared with patients without bone disease. Interestingly, circulating levels of miR-29b-3p correlated with cervical and thoracic vertebral lesions, while miR-195-5p correlated with thoracic lesions. Our findings suggest circulating miRNAs can be promising biomarkers for MM diagnosis and that their levels correlate with myeloma bone disease and osteolytic lesions.

Long non-coding RNAs (lncRNAs) are master regulators of gene expression and have recently emerged as potential innovative therapeutic targets. The deregulation of lncRNA expression patterns has been associated with age-related and noncommunicable diseases in the bone tissue, including osteoporosis and tumors. However, the specific role of lncRNAs in physiological or pathological conditions in the bone tissue still needs to be further clarified, for their exploitation as therapeutic tools. In the present study, we evaluate the potential of the lncRNA CASC2 as a regulator of osteogenic differentiation and mineralization. Results show that CASC2 expression is decreased during osteogenic differentiation of human bone marrow-derived Mesenchymal Stem/Stromal cells (hMSCs). CASC2 knockdown, using small interfering RNA against CASC2 (siCASC2), increases the expression of the late osteogenic marker Bone Sialoprotein (BSP), but does not impact ALP staining level nor the expression of early osteogenic transcripts, including RUNX2 and OPG. Although siCASC2 does not impact hMSC proliferation nor apoptosis, it promotes the mineralization of hMSC cultured under osteogenic-inducing conditions, as shown by the increase of calcium deposits. Mass spectrometry-based proteomic analysis revealed that 89 proteins are regulated by CASC2 at late osteogenic stages, including proteins associated with bone diseases or anthropometric and musculoskeletal traits. Specifically, the Cartilage Oligomeric Matrix Protein (COMP) is highly enhanced by CASC2 knockdown at late stages of osteogenic differentiation, at both transcriptional and protein level. On the other hand, inhibition of COMP impairs osteoblasts mineralization as well as the expression of BSP. The results indicate that lncRNA CASC2 regulates late osteogenic differentiation and mineralization in hMSC via COMP and BSP. In conclusion, this study suggests that targeting lncRNA CASC2 could be a potential approach for modulating bone mineralization.

Advanced therapy medicinal products (ATMPs) involving mesenchymal stromal/stem cells (MSCs) have emerged as a promising therapeutic approach for a wide spectrum of diseases, including several musculoskeletal disorders and bone fractures. Despite their potential, clinical translation has often led to inconsistent outcomes and the therapeutic efficacy of unmodified MSCs remains suboptimal. To overcome these challenges, strategies to enhance the functional properties of MSCs are needed. MicroRNAs (miRNAs) are master regulators of gene expression and have emerged as powerful tools for engineering MSCs to enhance their pro-regenerative capacity and potentially improve clinical outcomes. High-throughput RNA sequencing was performed to characterize transcriptional changes induced by anti-miR-195 modulation. Anti-miR-195-modulated MSCs were seeded onto collagen/hydroxyapatite scaffolds and their potential was evaluated in vivo using a heterotopic bone formation mouse model. Histological and molecular analyses were performed at 14 and 28 days post-implantation to assess cell migration, osteogenic marker expression, collagen deposition and vascularization. Anti-miR-195-modulated MSCs displayed transcriptional profiles associated with enhanced migration, invasion, and cell cycle progression, together with reduced senescence, cell death and apoptosis. Expression of the bone mineral density-associated transcript IBSP was significantly increased. In vivo, scaffolds seeded with anti-miR-195-treated MSCs showed increased cell migration at day 14 post-implantation. At day 28 post-implantation, enhanced collagen deposition and increased expression of osteogenic markers, including RUNX2 and SPP1, were observed. In addition, the scaffold area covered by blood vessels and the number of larger vessels were significantly higher in anti-miR-195-MSC scaffolds compared with controls. These findings demonstrate that anti-miR-195 promotes cell migration and vascularization in vivo, supporting miRNA-based engineering as a promising strategy to improve the efficacy of MSC-based ATMPs for musculoskeletal diseases and bone defects.

Purpose of Review Circular RNAs (circRNAs) are RNA transcripts derived from fragments of pre-messenger RNAs through a back-splicing process. An advantage that rises from their circular covalently closed conformation is their high stability, when compared with their linear counterparts. The current review focuses on the emerging roles of circRNAs in osteoporosis, including in osteogenic differentiation and osteoclastogenesis. Their potential as osteoporosis biomarkers will also be discussed. Recent Findings Although firstly described as non-coding, some of these single-stranded RNAs were recently reported to possess protein-coding capacity. On the other hand, the circRNAs exhibit cell and tissue-specific patterns at the transcriptome level in eukaryotes and are regulated throughout the development or disease progression. Even though thousands of these circular transcripts are listed and annotated, only a limited number of studies describe their biological role in bone processes. Recent evidence indicates inhibitory activator roles in both osteoblasts and osteoclasts differentiation and function. Latest screenings in the blood, plasma, or serum of osteoporosis patients support the potential for circRNA signature to be used as biomarkers in osteoporosis, but further validation is required. Summary While intense research into circRNAs has been detailing their biological roles, there remains a need for standardization and further research to fulfil the future potential of this emerging and highly promising class of regulatory molecules.